. POLYMERAGE CHAIN REACTION (PCR)

POLYMERAGE CHAIN REACTION (PCR)

   a. INTRODUCTION

The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique for exponentially amplifying DNA via enzymatic replication, without using a living organism (such as E. coli or yeast).

PCR was conceived by American biochemist Kari B. Mullis in 1983 and was later developed by Mullis and his associate Fred A. Felon at the Cletus Corporation in Emeryville, California. Although the value of PCR was not immediately recognized, by 1991 its use had become widespread. For his work, Mullis was named a cowinner of the 1993 Nobel Prize in chemistry.

PCR proceeds in a series of cycles, or rounds. Each successive round doubles the amount of DNA and thus more than 1 billion copies of a single DNA fragment can be made in just a few hours. The technique of PCR is simple enough to be used by scientists with little training in molecular biology. The supplies necessary for carrying out PCR are available in a kit form that is used in such varied settings as crime laboratories and clinical diagnostic laboratories.

 b. COMPONENTS REQUIRED FOR PCR

Thermal cycler

Template DNA

PCR buffer

Taq polymerase(enzyme)

DNTP

Mgcl2(to make enzyme work)

Primer-1(Forward)

Primer-2(reverse)

 c. LABORATORY REQUIREMENTS

Items for preparing and pipetting solutions (PH meter, pipettes, stirrers, glassware etc). Electrophoresis chambers; power supply; micro centrifuge; ultravioletcamera; a source deionized water and acess to an autoclave.

Different Types of PCR

Touchdown PCR

Hot-start PCR

Multiplex PCR

Assembly PCR                                                                 

Quantitative PCR

Colony PCR

Reverse Transcription PCR

Asymmetric PCR

 d. PROCESSES OF PCR

Polymerase chain reaction (PCR) uses an enzyme known as polymerase to rapidly multiply a small fragment of deoxyribonucleic acid , ladderlike molecule that carries the hereditary material in all living things. Each cycle of PCR consists of three phases. In the first phase, denaturation, the DNA is heated to cause its two linked strands to separate. In the second phase, annealing, the temperature of the mixture is lowered to allow primers—starter pieces of DNA—to bind to the separated DNA. In the third phase, polymerization, the temperature is raised to allow the polymerase enzyme to rapidly copy the DNA. Each PCR cycle duplicates the existing DNA, so over 1 billion copies of a single DNA fragment can be made in just a few hours.

The polymerase chain reaction mimics the DNA replication, or reproduction, process that occurs naturally in living cells. Most DNA is double-stranded—that is, each strand of DNA is paired with a complementary strand. During replication, the two strands of DNA separate and a specialized cell enzyme called polymerase makes a copy of each strand, using the original strand as a template, or pattern. Normally this copying occurs when cells divide and results in the production of one pair of daughter strands for each of the two parent strands.

Polymerase requires two additional ingredients to copy DNA. The first is a supply of the four basic building blocks of DNA, called nucleotide bases. The second is a short stretch of copied DNA, called an oligonucleotide primer, consisting of several nucleotides that initiate replication. PCR uses these same ingredients to copy DNA in a vial.

There are three phases in a polymerase chain reaction. In the first phase, called denaturation, the template, or piece of original DNA, is heated to a temperature of from 90º to 95º C for 30 seconds, which causes the individual strands to separate. In the second phase, called annealing, the temperature of the mixture is lowered to 55º C over a 20-second period, allowing the oligonucleotide primers to bind to the separated DNA. In the third phase, called polymerization, the temperature of the mixture is raised to 75º C, a temperature at which the polymerase can copy the DNA molecule rapidly.

These three phases are carried out in the same vial and make up one complete PCR cycle, which takes less than two minutes to complete. Theoretically, the PCR cycle can be repeated indefinitely, but the polymerase, nucleotides, and primers are usually renewed after about 30 cycles. Thirty PCR cycles can produce 1 billion DNA copies in less than three hours.

The polymerase used in early PCR procedures was easily destroyed by heat. Consequently, additional polymerase had to be added in each PCR cycle to replace that destroyed by the high temperatures of the first phase. In modern PCR procedures, however, the heat-stable Taq polymerase is usually used. It was originally extracted from Thermus aquaticus, a heat-loving bacterium found in the hot springs of Yellowstone National Park. Because Taq polymerase is not destroyed by the high temperatures of PCR, it is only necessary to add it once, at the beginning of the reaction. Taq polymerase is now produced for PCR by genetically engineered bacteria.

The use of PCR requires great care. The chief concern is contamination of the reaction mix. PCR is so sensitive that it is possible to accidentally multiply minute amounts of contaminating DNA. Special procedures are used to ensure that such contamination is avoided.                                           

  e. USES OF PCR

Because only minute amounts of relatively crude DNA samples are required for PCR, it is a valuable tool for research in biology, clinical medicine, and forensic science.

In biological research, PCR has accelerated the study of gene function, gene mapping, and evolution. Gene-function research uses PCR to create copies of individual genes, the activities of which can then be studied and more precisely defined. Gene mapping relies on PCR to create many copies of specific regions of human DNA. These regions can then be examined to see if they are linked to genetic diseases, such as cystic fibrosis.

The study of evolution has also benefited from PCR. For example, scientists have used PCR to study DNA from insects trapped for thousands of years in amber. Even though much of the DNA has lost its structure, there is still enough intact DNA remaining to be multiplied by PCR for comparison with the DNA of present-day insects.

In medicine, PCR is particularly useful in prenatal testing for genetic diseases. DNA samples obtained from a fetus by amniocentesis, in which a small sample of fluid is drawn from the mother's uterus, can be tested by PCR in just a few hours. Previously, it was necessary to culture fetal cells, or grows them in a special nutrient medium, for several weeks before biochemical tests could be performed. PCR testing has also been used on cells taken from hours-old embryos fertilized in vitro to identify an embryo that was free of disease. The embryo was then implanted in the mother's uterus, and a normal pregnancy resulted.

Other medical applications of PCR include identifying viruses, bacteria, and cancerous cells in human tissues. PCR can even be used within single cells, in a procedure called in situ PCR, to identify specific cell types.

In forensic science, PCR has revolutionized the process of criminal identification. PCR-based DNA-typing tests can create detailed DNA fingerprints that can definitively identify individuals. Such tests can also exclude or implicate suspects based on small amounts of blood, skin, hair, or semen left at a crime scene.

PCR has been used to trace industrial waste and other products. Small amounts of known DNA are inserted into batches of explosives, petroleum products, poisons, and other waste at their source to create a tag that will last indefinitely. Such DNA tags can be recovered from oil slicks or other pollutants found in public waterways for example, and then multiplied by PCR and compared with a listing of manufacturers' DNA tags. A match can provide strong evidence implicating polluters.

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